Evidence from simian virus 40 (SV40) cell free DNA replication studies indicates that the DNA polymerase alpha/primase complex is responsible for synthesis of the nascent DNA fragment during initiation of DNA replication, and for lagging strand DNA synthesis during elongation. Biochemical studies from viral and cellular systems both demonstrate that interactions between DNA polymerase alpha and other cellular replication proteins and cell growth relevant proteins are highly stringent and species specific. There are two major long term goals of our research. One is to understand the nature and regulation of DNA polymerase alpha interactions with other proteins during cell growth and at different stages of the cell cycle. The other is to define structure-function relationships of DNA polymerase alpha with its substrates. Our present proposal is a continuation of work already in progress in our laboratory.
The specific aims are as follows: A. Studies of the interaction of polymerase alpha with other proteins: (1) Identify domain(s) of polymerase alpha catalytic polypeptide that interacts with SV40 large T antigen. (2) Isolate cellular protein(s) that are the functional equivalent of T antigen using the above identified polymerase alpha protein domain(s) as affinity probe(s). (3) Develop affinity methods using the overproduced recombinant human DNA polymerase alpha as affinity ligand to isolate and characterize proteins/enzymes associated with DNA polymerase alpha at different stages of the cell cycle. B. Studies of the substrate interaction sites: (1) Define the domain(s) that interacts with DNA template by controlled proteolysis of polymerase alpha-DNA complex. (2) Perform site-directed mutagenesis of residues potentially critical for substrate binding and catalysis. (3) Characterize the enzymological properties such as kinetic parameters, DNA synthetic processivity and fidelity of the mutants. Knowledge gained from these studies will provide details of the molecular mechanisms of polymerase alpha catalysis, and will provide fundamental understanding of the molecular etiology of neoplasia.
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